Salinomycin

Vero cells were cultured in DMEM supplemented with 10% fetal bovine serum (FBS) and maintained at 37 °C in a humidified 5% CO₂ incubator. The PEDV live-attenuated strain Zhejiang08 was preserved in our laboratory [14 (link)]. Salinomycin was purchased from Selleck Chemicals (Houston, USA), and diluted to 10 mM in DMSO. Antibodies against phospho-p44/42 MAPK (Erk1/2) (Thr202/Tyr204), Erk1/2, phospho-p38MAPK (Thr180/Tyr182), p38MAPK, phospho-JNK((Thr183/Tyr185), JNK, GAPDH and HRP labeled secondary antibodies, including goat anti-mouse IgG and goat anti-rabbit IgG, were sourced from Cell Signaling Technology (Beverly, MA, USA).

HO-3867 (cat no: S7501), DFO (cat no: S6849), Dp44mT (cat no: S790), dexrazoxane HCl (cat no: S5651), salinomycin (cat no: S8129), ebselen (cat no: S6676), z.VAD.FMK (cat no: S7023), Nec-1 (cat no: S8037), Fer-1 (cat no: S7243), Lip-1 (cat no: S7699), and PFTα (cat no: S2929) were procured from Selleck Chemicals (USA). S-Benzylisothiourea (cat no: S9138) was ordered from Sigma-Aldrich (USA). The primary antibodies listed below were ordered from Cellular Signaling Technology (USA): caspase-3 (cat no:9662), Mcl-1 (cat no:94296), Bcl-2 (cat no:15071), Bcl-xL (cat no:2762), Bak (cat no:6947), Bax (cat no:94296), and DMT1 (cat no:15083). The following primary antibodies listed were procured from Abcam (USA): FPN1 (cat no: ab239583), TFR (cat no: ab214039), FTL (cat no: ab75973), FTH (cat no: ab183781), and p53 (cat no: ab26). The secondary antibody was procured from Abclonal (China). Sigma-Aldrich supplied all other routine chemicals.

BCLs were continuously treated for 72 h in adherent conditions with bortezomib (stock concentration SC = [10 mM], Selleckchem), carfilzomib (SC = [10 mM], Selleckchem), chloroquine (SC = [10 mM], Selleckchem), Cortistatin A (SC = [100 μM], a kind gift from Prof. P. Baran, The Scripps Research Institute, La Jolla, CA, USA), docetaxel (SC = [10 mM], GSK343 (SC = [1 mM], Active Biochemicals Co), I‐CBP112 (SC = [10 mM], Sigma), JQ1 (SC = [10 mM], Selleckchem), mifepristone (SC = [10 mM], Selleckchem), Ro5‐3335 (SC = [10 mM], Calbiochem), ruxolitinib (SC = [10 mM], Selleckchem), SGC‐CBP30 (SC = [10 mM], Selleckchem), salinomycin (SC = [200 μM], Selleckchem), spliceostatin A (SC = [10 mM], Adooq Biosciences), tazemetostat (SC = [5 mM], Selleckchem), and 8‐AZA (SC = [100 mM], Chembiotech). All these compounds were resuspended in dimethyl sulfoxide (DMSO; Sigma), except for chloroquine resuspended in H₂O. For the in vivo experiments, salinomycin (SC = [6 mg/ml], Medchemexpress) and JQ1 (SC = [100 mg/ml], Medchemexpress) were resuspended in a solution of DMSO/(2‐Hydroxypropyl)‐β‐cyclodextrin (HPCD) 10% (1:9, v/v).

DU-145 CRPC cells (ATCC) and NCI-H660 NEPC cells (ATCC) were cultured in RPMI1640 medium (Thermo Fisher Scientific, Waltham, MA, USA) supplemented with 10% FBS. BT-549 TNBC cells (ATCC) were cultured in RPMI1640 medium containing 10% FBS and 10 μg/mL insulin. MDA-MB-468 TNBC cells were cultured in Leibovitz’s L-15 Medium (Thermo Fisher Scientific) supplemented with 10% FBS. Cells were treated with the MUC1-C inhibitor GO-203 [3 (link), 4 (link), 48 ], salinomycin (S8129, SelleckChem, Houston, TX, USA), BAY11-7082 (SelleckChem) and salinomycin encapsulated in polymeric nanoparticles (SAL/NPs, HSB-1216; HillstreamBiopharma, Bridgewater, NJ, USA). Cell viability was assessed using the Alamar Blue assay (Thermo Scientific, Rockford, IL, USA) in sextuplicate wells. The IC50 value was determined by nonlinear regression of the dose–response data using Prism 9.0 (GraphPad Software). Authentication of the cells was performed by short tandem repeat (STR) analysis. Cells were monitored for mycoplasma contamination using the MycoAlert Mycoplasma Detection Kit (Lonza, Rockland, MA, USA). Cells were maintained for 3 months for performing experiments.

Cells were seeded in multiwell plates and treated with the indicated concentrations of salinomycin (Selleck Chemicals, Munich, Germany) for 72 hours. Cells were harvested, washed in PBS and resuspended in PBS containing 1% bovine serum albumin. The same antibodies, concentrations and incubation times as for immunofluorescence staining were used. After several washing steps, cells were analyzed by a BD FACS Calibur Flow Cytometer (Becton Dickinson).